Pioglitazone attenuates valvular calcification induced by hypercholesterolemia

Abstract

Objective: Development of calcific aortic valve stenosis involves multiple signaling pathways, which may be modulated by peroxisome proliferator-activated receptor-γ). This study tested the hypothesis that pioglitazone (Pio), a ligand for peroxisome proliferator-activated receptor-γ, inhibits calcification of the aortic valve in hypercholesteremic mice.

Methods and results: Low density lipoprotein receptor(-/-)/apolipoprotein B(100/100) mice were fed a Western-type diet with or without Pio (20 mg/kg per day) for 6 months. Pio attenuated lipid deposition and calcification in the aortic valve, but not aorta. In the aortic valve, Pio reduced levels of active caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Valve function (echocardiography) was significantly improved by Pio. To determine whether changes in gene expression are associated with differential effects of Pio on aortic valves versus aorta, Reversa mice were fed Western diet with or without Pio for 2 months. Several procalcific genes were increased by Western diet, and the increase was attenuated by Pio, in aortic valve, but not aorta.

Conclusions: Pio attenuates lipid deposition, calcification, and apoptosis in aortic valves of hypercholesterolemic mice, improves aortic valve function, and exhibits preferential effects on aortic valves versus aorta. We suggest that Pio protects against calcific aortic valve stenosis, and Pio or other peroxisome proliferator-activated receptor-γ ligands may be useful for early intervention to prevent or slow stenosis of aortic valves.

Figures

Figure 1

A. Summary of experimental protocol. 1B. Effects of pioglitazone on calcification of aortic valve and aorta of LA mice. Calcification of the aortic valve and ascending aorta was measured as percent of area stained with Alizarin Red in the base of aortic valves and the ascending aorta. Pioglitazone attenuated calcification of the aortic valve, but not ascending aorta, produced by Western diet in LA mice. n=9-13. 1C. Effects of pioglitazone on lipid deposition in aortic valve and ascending aorta of LA mice. Lipid deposition was measured as percent of area stained with Oil Red O. Pioglitazone attenuated lipid deposition produced by Western diet in aortic valves, but not in the aorta. n=5-8. 1D. Effects of pioglitazone on collagen in aortic valve and ascending aorta of LA mice. Collagen was measured as percent of area stained with Masson's staining. Pioglitazone, nor Western diet, had an effect on collagen. n=6-8. Values are mean±SE, * =p <0.05 vs. chow, ** = p<0.05 vs. WD.

Figure 2

A. Effects of pioglitazone on protein expression of osterix and osteocalcin in aortic valves of LA mice. Protein expression was measured as percent of area stained with antibody. Osterix and osteocalcin were not significantly different among the three groups. n=7-10. 2B. Effects of pioglitazone on protein expression of active caspase-3 in aortic valves and aorta of LA mice. Protein expression was measured as percent of area stained with antibody. Pioglitazone prevented the increase in expression of active caspase-3 produced by Western diet. n=7-10. Values are mean±SE, * = p<0.05 vs. chow; **=p<0.05 vs. WD. 2C. Colocalization (yellow) of active caspase-3 (red) and cell markers (green). Active caspase-3 is colocalized in aortic valves with myofibroblast (α-smooth muscle actin-positive), macrophage (F4/80-positive), and osteoblast-like cells (Cbfa1-positve).

Figure 2

A. Effects of pioglitazone on protein expression of osterix and osteocalcin in aortic valves of LA mice. Protein expression was measured as percent of area stained with antibody. Osterix and osteocalcin were not significantly different among the three groups. n=7-10. 2B. Effects of pioglitazone on protein expression of active caspase-3 in aortic valves and aorta of LA mice. Protein expression was measured as percent of area stained with antibody. Pioglitazone prevented the increase in expression of active caspase-3 produced by Western diet. n=7-10. Values are mean±SE, * = p<0.05 vs. chow; **=p<0.05 vs. WD. 2C. Colocalization (yellow) of active caspase-3 (red) and cell markers (green). Active caspase-3 is colocalized in aortic valves with myofibroblast (α-smooth muscle actin-positive), macrophage (F4/80-positive), and osteoblast-like cells (Cbfa1-positve).

Figure 3

Effects of pioglitazone on mRNA expression in aortic valves and aorta of Reversa mice at 14 months of age. During the last 2 months, mice were fed WD or WD+Pio. The expression pattern of Rbp7 and osteocalcin was similar in aortic valves and the aorta, whereas IL-6, Cbfa1, and collagen 1a2 exhibited dissimilar patterns between aortic valves and the aorta. Values are mean±SE in 11-12 mice for aortic valves, and 8-9 for the aorta. * = p

Figure 4

Effects of pioglitazone on separation distance of aortic valve cusps during systole of LA mice. Values were obtained in LA mice fed chow, Western diet (WD) without and with pioglitazone (pio) (20 mg/kg/day) for 6 months. Values are mean±SE in 5-6 mice per group. ** = p